Late adolescence mortality in mice with brain-specific deletion of the volume-regulated anion channel subunit LRRC8A.

The leucine-rich repeat-containing family 8 member A (LRRC8A) is an essential subunit of the volume-regulated anion channel (VRAC). VRAC is critical for cell volume control, but its broader physiological functions remain under investigation. Recent studies in the field indicate that Lrrc8a disruption in the brain astrocytes reduces neuronal excitability, impairs synaptic plasticity and memory, and protects against cerebral ischemia. In the present work, we generated brain-wide conditional LRRC8A knockout mice (LRRC8A bKO) using NestinCre -driven Lrrc8aflox/flox excision in neurons, astrocytes, and oligodendroglia. LRRC8A bKO animals were born close to the expected Mendelian ratio and developed without overt histological abnormalities, but, surprisingly, all died between 5 and 9 weeks of age with a seizure phenotype, which was confirmed by video and EEG recordings. Brain slice electrophysiology detected changes in the excitability of pyramidal cells and modified GABAergic inputs in the hippocampal CA1 region of LRRC8A bKO. LRRC8A-null hippocampi showed increased immunoreactivity of the astrocytic marker GFAP, indicating reactive astrogliosis. We also found decreased whole-brain protein levels of the GABA transporter GAT-1, the glutamate transporter GLT-1, and the astrocytic enzyme glutamine synthetase. Complementary HPLC assays identified reduction in the tissue levels of the glutamate and GABA precursor glutamine. Together, these findings suggest that VRAC provides vital control of brain excitability in mouse adolescence. VRAC deletion leads to a lethal phenotype involving progressive astrogliosis and dysregulation of astrocytic uptake and supply of amino acid neurotransmitters and their precursors.

Eight Flurothyl-Induced Generalized Seizures Lead to the Rapid Evolution of Spontaneous Seizures in Mice: A Model of Epileptogenesis with Seizure Remission.

UNLABELLED: The occurrence of recurrent, unprovoked seizures is the hallmark of human epilepsy. Currently, only two-thirds of this patient population has adequate seizure control. New epilepsy models provide the potential for not only understanding the development of spontaneous seizures, but also for testing new strategies to treat this disorder. Here, we characterize a primary generalized seizure model of epilepsy following repeated exposure to the GABAA receptor antagonist, flurothyl, in which mice develop spontaneous seizures that remit within 1 month. In this model, we expose C57BL/6J mice to flurothyl until they experience a generalized seizure. Each of these generalized seizures typically lasts <30 s. We induce one seizure per day for 8 d followed by 24 h video-electroencephalographic recordings. Within 1 d following the last of eight flurothyl-induced seizures, ∼50% of mice have spontaneous seizures. Ninety-five percent of mice tested have seizures within the first week of the recording period. Of the spontaneous seizures recorded, the majority are generalized clonic seizures, with the remaining 7-12% comprising generalized clonic seizures that transition into brainstem seizures. Over the course of an 8 week recording period, spontaneous seizure episodes remit after ∼4 weeks. Overall, the repeated flurothyl paradigm is a model of epileptogenesis with spontaneous seizures that remit. This model provides an additional tool in our armamentarium for understanding the mechanisms underlying epileptogenesis and may provide insights into why spontaneous seizures remit without anticonvulsant treatment. Elucidating these processes could lead to the development of new epilepsy therapeutics. SIGNIFICANCE STATEMENT: Epilepsy is a chronic disorder characterized by the occurrence of recurrent, unprovoked seizures in which the individual seizure-ictal events are self-limiting. Remission of recurrent, unprovoked seizures can be achieved in two-thirds of cases by treatment with anticonvulsant medication, surgical resection, and/or nerve/brain electrode stimulation. However, there are examples in humans of epilepsy with recurrent, unprovoked seizures remitting without any intervention. While elucidating how recurrent, unprovoked seizures develop is critical for understanding epileptogenesis, an understanding of how and why recurrent, unprovoked seizures remit may further our understanding and treatment of epilepsy. Here, we describe a new model of recurrent, unprovoked spontaneous seizures in which the occurrence of spontaneous seizures naturally remits over time without any therapeutic intervention.

Opioid Analgesia in P450 Gene Cluster Knockout Mice: A Search for Analgesia-Relevant Isoforms.

Cytochrome P450 monooxygenases (P450s), which are well-known drug-metabolizing enzymes, are thought to play a signal transduction role in µ opioid analgesia and may serve as high-affinity (3)H-cimetidine ((3)HCIM) binding sites in the brain. (3)HCIM binding sites may also be related to opioid or nonopioid analgesia. However, of the more than 100 murine P450 enzymes, the specific isoform(s) responsible for either function have not been identified. Presently, three lines of constitutive P450 gene cluster knockout (KO) mice with full-length deletions of 14 Cyp2c, 9 Cyp2d, and 7 Cyp3a genes were studied for deficiencies in (3)HCIM binding and for opioid analgesia. Liver and brain homogenates from all three genotypes showed normal (3)HCIM binding values, indicating that gene products of Cyp2d, Cyp3a, and Cyp2c are not (3)HCIM-binding proteins. Cyp2d KO and Cyp3a KO mice showed normal antinociceptive responses to a moderate systemic dose of morphine (20 mg/kg, s.c.), thereby excluding 16 P450 isoforms as mediators of opioid analgesia. In contrast, Cyp2c KO mice showed a 41% reduction in analgesic responses following systemically (s.c.) administered morphine. However, the significance of brain Cyp2c gene products in opioid analgesia is uncertain because little or no analgesic deficits were noted in Cyp2c KO mice following intracerebroventricular or intrathecalmorphine administration, respectively. These results show that the gene products of Cyp2d and Cyp3a do not contribute to µ opioid analgesia in the central nervous system. A possible role for Cyp2c gene products in opioid analgesia requires further consideration.

Conditional deletion of LRRC8A in the brain reduces stroke damage independently of swelling-activated glutamate release.

The ubiquitous volume-regulated anion channels (VRACs) facilitate cell volume control and contribute to many other physiological processes. Treatment with non-specific VRAC blockers or brain-specific deletion of the essential VRAC subunit LRRC8A is highly protective in rodent models of stroke. Here, we tested the widely accepted idea that the harmful effects of VRACs are mediated by release of the excitatory neurotransmitter glutamate. We produced conditional LRRC8A knockout either exclusively in astrocytes or in the majority of brain cells. Genetically modified mice were subjected to an experimental stroke (middle cerebral artery occlusion). The astrocytic LRRC8A knockout yielded no protection. Conversely, the brain-wide LRRC8A deletion strongly reduced cerebral infarction in both heterozygous (Het) and full KO mice. Yet, despite identical protection, Het mice had full swelling-activated glutamate release, whereas KO animals showed its virtual absence. These findings suggest that LRRC8A contributes to ischemic brain injury via a mechanism other than VRAC-mediated glutamate release.

Pain-facilitating medullary neurons contribute to opioid-induced respiratory depression.

Respiratory depression is a therapy-limiting side effect of opioid analgesics, yet our understanding of the brain circuits mediating this potentially lethal outcome remains incomplete. Here we studied the contribution of the rostral ventromedial medulla (RVM), a region long implicated in pain modulation and homeostatic regulation, to opioid-induced respiratory depression. Microinjection of the µ-opioid agonist DAMGO in the RVM of lightly anesthetized rats produced both analgesia and respiratory depression, showing that neurons in this region can modulate breathing. Blocking opioid action in the RVM by microinjecting the opioid antagonist naltrexone reversed the analgesic and respiratory effects of systemically administered morphine, showing that this region plays a role in both the analgesic and respiratory-depressant properties of systemically administered morphine. The distribution of neurons directly inhibited by RVM opioid microinjection was determined with a fluorescent opioid peptide, dermorphin-Alexa 594, and found to be concentrated in and around the RVM. The non-opioid analgesic improgan, like DAMGO, produced antinociception but, unlike DAMGO, stimulated breathing when microinjected into the RVM. Concurrent recording of RVM neurons during improgan microinjection showed that this agent activated RVM ON-cells, OFF-cells, and NEUTRAL-cells. Since opioids are known to activate OFF-cells but suppress ON-cell firing, the differential respiratory response to these two analgesic drugs is best explained by their opposing effects on the activity of RVM ON-cells. These findings show that pain relief can be separated pharmacologically from respiratory depression and identify RVM OFF-cells as important central targets for continued development of potent analgesics with fewer side effects.

Physiological basis for inhibition of morphine and improgan antinociception by CC12, a P450 epoxygenase inhibitor.

Many analgesic drugs, including µ-opioids, cannabinoids, and the novel nonopioid analgesic improgan, produce antinociception by actions in the rostral ventromedial medulla (RVM). There they activate pain-inhibiting neurons, termed "OFF-cells," defined by a nociceptive reflex-related pause in activity. Based on recent functional evidence that neuronal P450 epoxygenases are important for the central antinociceptive actions of morphine and improgan, we explored the convergence of opioid and nonopioid analgesic drug actions in RVM by studying the effects of the P450 epoxygenase inhibitor CC12 on the analgesic drug-induced activation of these OFF-cells and on behavioral antinociception. In rats lightly anesthetized with isoflurane, we recorded the effects of intraventricular morphine and improgan, with and without CC12 pretreatment, on tail flick latency and activity of identified RVM neurons: OFF-cells, ON-cells (pronociceptive neurons), and neutral cells (unresponsive to analgesic drugs). CC12 pretreatment preserved reflex-related changes in OFF-cell firing and blocked the analgesic actions of both drugs, without interfering with the increase in spontaneous firing induced by improgan or morphine. CC12 blocked suppression of evoked ON-cell firing by improgan, but not morphine. CC12 pretreatment had no effect by itself on RVM neurons or behavior. These data show that the epoxygenase inhibitor CC12 works downstream from receptors for both µ-opioid and improgan, at the inhibitory input mediating the OFF-cell pause. This circuit-level analysis thus provides a cellular basis for the convergence of opioid and nonopioid analgesic actions in the RVM. A presynaptic P450 epoxygenase may therefore be an important target for development of clinically useful nonopioid analgesic drugs.

High-affinity binding of [3H]cimetidine to a heme-containing protein in rat brain.

[(3)H]Cimetidine (3HCIM) specifically binds to an unidentified site in the rat brain. Because recently described ligands for this site have pharmacological activity, 3HCIM binding was characterized. 3HCIM binding was saturable, heat-labile, and distinct from the histamine H(2) receptor. To test the hypothesis that 3HCIM binds to a cytochrome P450 (P450), the effects of nonselective and isoform-selective P450 inhibitors were studied. The heme inhibitor KCN and the nonselective P450 inhibitor metyrapone both produced complete, concentration-dependent inhibition of 3HCIM binding (K(i) = 1.3 mM and 11.9 muM, respectively). Binding was largely unaffected by inhibitors of CYP1A2, 2B6, 2C8, 2C9, 2D6, 2E1, and 19A1 but was eliminated by inhibitors of CYP2C19 (tranylcypromine) and CYP3A4 (ketoconazole). Synthesis and testing of CC11 [4(5)-(benzylthiomethyl)-1H-imidazole] and CC12 [4(5)-((4-iodobenzyl)-thiomethyl)-1H-imidazole] confirmed both drugs to be high-affinity inhibitors of 3HCIM binding. On recombinant human P450s, CC12 was a potent inhibitor of CYP2B6 (IC(50) = 11.7 nM), CYP2C19 (51.4 nM), and CYP19A1 (140.7 nM) and had a range of activities (100-494 nM) on nine other isoforms. Although the 3HCIM binding site pharmacologically resembles some P450s, eight recombinant human P450s and three recombinant rat P450s did not exhibit 3HCIM binding. Inhibition by KCN and metyrapone suggests that 3HCIM binds to a heme-containing brain protein (possibly a P450). However, results with selective P450 inhibitors, recombinant P450 isoforms, and a P450 antibody did not identify a 3HCIM-binding P450 isoform. Finally, CC12 is a new, potent inhibitor of CYP2B6 and CYP2C19 that may be a valuable tool for P450 research.

BranchAnalysis2D/3D automates morphometry analyses of branching structures.

BACKGROUND: Morphometric analyses of biological features have become increasingly common in recent years with such analyses being subject to a large degree of observer bias, variability, and time consumption. While commercial software packages exist to perform these analyses, they are expensive, require extensive user training, and are usually dependent on the observer tracing the morphology. NEW METHOD: To address these issues, we have developed a broadly applicable, no-cost ImageJ plugin we call 'BranchAnalysis2D/3D', to perform morphometric analyses of structures with branching morphologies, such as neuronal dendritic spines, vascular morphology, and primary cilia. RESULTS: Our BranchAnalysis2D/3D algorithm allows for rapid quantification of the length and thickness of branching morphologies, independent of user tracing, in both 2D and 3D data sets. COMPARISON WITH EXISTING METHODS: We validated the performance of BranchAnalysis2D/3D against pre-existing software packages using trained human observers and images from brain and retina. We found that the BranchAnalysis2D/3D algorithm outputs results similar to available software (i.e., Metamorph, AngioTool, Neurolucida), while allowing faster analysis times and unbiased quantification. CONCLUSIONS: BranchAnalysis2D/3D allows inexperienced observers to output results like a trained observer but more efficiently, thereby increasing the consistency, speed, and reliability of morphometric analyses.

CC12, a high-affinity ligand for [3H]cimetidine binding, is an improgan antagonist.

Improgan, a chemical congener of cimetidine, is a highly effective non-opioid analgesic when injected into the CNS. Despite extensive characterization, neither the improgan receptor, nor a pharmacological antagonist of improgan has been previously described. Presently, the specific binding of [(3)H]cimetidine (3HCIM) in brain fractions was used to discover 4(5)-((4-iodobenzyl)thiomethyl)-1H-imidazole, which behaved in vivo as the first improgan antagonist. The synthesis and pharmacological properties of this drug (named CC12) are described herein. In rats, CC12 (50-500nmol, i.c.v.) produced dose-dependent inhibition of improgan (200-400nmol) antinociception on the tail flick and hot plate tests. When given alone to rats, CC12 had no effects on nociceptive latencies, or on other observable behavioral or motor functions. Maximal inhibitory effects of CC12 (500nmol) were fully surmounted with a large i.c.v. dose of improgan (800nmol), demonstrating competitive antagonism. In mice, CC12 (200-400nmol, i.c.v.) behaved as a partial agonist, producing incomplete improgan antagonism, but also limited antinociception when given alone. Radioligand binding, receptor autoradiography, and electrophysiology experiments showed that CC12's antagonist properties are not explained by activity at 25 sites relevant to analgesia, including known receptors for cannabinoids, opioids or histamine. The use of CC12 as an improgan antagonist will facilitate the characterization of improgan analgesia. Furthermore, because CC12 was also found presently to inhibit opioid and cannabinoid antinociception, it is suggested that this drug modifies a biochemical mechanism shared by several classes of analgesics. Elucidation of this mechanism will enhance understanding of the biochemistry of pain relief.

Improgan-induced hypothermia: a role for cannabinoid receptors in improgan-induced changes in nociceptive threshold and body temperature.

Improgan, a congener of the H(2) antagonist cimetidine, produces non-opioid antinociception which is blocked by the CB(1) antagonist rimonabant, implying a cannabinoid mechanism of action. Since cannabinoids produce hypothermia as well as antinociception in rodents, the present study investigated the pharmacological activity of improgan on core body temperature and nociceptive (tail flick) responses. Improgan (60, 100 and 140 microg, intraventricular [ivt]) elicited significant decreases in core temperature 3-30 min following injection with a maximal hypothermic effect of -1.3 degrees C. Pretreatment with rimonabant (50 microg, ivt) produced a statistically significant but incomplete (29-42%) antagonism of improgan hypothermia. In control experiments, the CB(1) agonist CP-55,940 (37.9 microg, ivt) induced significant decreases in core temperature (-1.8 degrees C) 3-30 min following injection. However, unlike the case with improgan, pretreatment with rimonabant completely blocked CP-55,940 hypothermia. Furthermore, CP-55,940 and improgan elicited maximal antinociception over the same time course and dose ranges, and both effects were attenuated by rimonabant. These results show that, like cannabinoid agonists in the rat, improgan produces antinociception and hypothermia which is blocked by a CB(1) antagonist. Unlike cannabinoid agonists, however, improgan does not produce locomotor inhibition at antinociceptive doses. Additional experiments were performed to determine the effect of CC12, a recently discovered improgan antagonist which lacks affinity at CB(1) receptors. Pretreatment with CC12 (183 microg, ivt) produced complete inhibition of both the antinociception and the hypothermia produced by improgan, suggesting the possible role of an unknown improgan receptor in both of these effects.

Significance of cannabinoid CB1 receptors in improgan antinociception.

UNLABELLED: Improgan is a congener of the H(2) antagonist cimetidine, which produces potent antinociception. Because a) the mechanism of action of improgan remains unknown and b) this drug may indirectly activate cannabinoid CB(1) receptors, the effects of the CB(1) antagonist/inverse agonist rimonabant (SR141716A) and 3 congeners with varying CB(1) potencies were studied on improgan antinociception after intracerebroventricular (icv) dosing in rats. Consistent with blockade of brain CB(1) receptors, rimonabant (K(d) = 0.23 nM), and O-1691 (K(d) = 0.22 nM) inhibited improgan antinociception by 48% and 70% after icv doses of 43 nmol and 25 nmol, respectively. However, 2 other derivatives with much lower CB(1) affinity (O-1876, K(d) = 139 nM and O-848, K(d) = 352 nM) unexpectedly blocked improgan antinociception by 65% and 50% after icv doses of 300 nmol and 30 nmol, respectively. These derivatives have 600-fold to 1500-fold lower CB(1) potencies than that of rimonabant, yet they retained improgan antagonist activity in vivo. In vitro dose-response curves with (35)S-GTPgammaS on CB(1) receptor-containing membranes confirmed the approximate relative potency of the derivatives at the CB(1) receptor. Although antagonism of improgan antinociception by rimonabant has previously implicated a mechanistic role for the CB(1) receptor, current findings with rimonabant congeners suggest that receptors other than, or in addition to CB(1) may participate in the pain-relieving mechanisms activated by this drug. The use of congeners such as O-848, which lack relevant CB(1)-blocking properties, will help to identify these cannabinoid-like, non-CB(1) mechanisms. PERSPECTIVE: This article describes new pharmacological characteristics of improgan, a pain-relieving drug that acts by an unknown mechanism. Improgan may use a marijuana-like (cannabinoid) pain-relieving mechanism, but it is shown presently that the principal cannabinoid receptor in the brain (CB(1)) is not solely responsible for improgan analgesia.

Antinociceptive activity of chemical congeners of improgan: optimization of side chain length leads to the discovery of a new, potent, non-opioid analgesic.

Improgan is a chemical congener of the H2 antagonist cimetidine which shows the profile of a highly effective analgesic when administered directly into the CNS. Although the improgan receptor is unknown, improgan activates analgesic pathways which are independent of opioids, but may utilize cannabinoid mechanisms. To discover selective, potent, improgan-like drugs, seven compounds chemically related to improgan were synthesized and tested for antinociceptive activity in rats after intracerebroventricular (icv) administration. Among a series of improgan congeners in which the alkyl chain length of improgan ((-CH2)3-) was varied, five compounds showed full agonist antinociceptive activity with potencies greater than that of improgan. VUF5420 (containing (-CH2)4-, EC50 = 86.1 nmol) produced maximal antinociceptive activity after doses which showed no motor impairment or other obvious toxicity, and was 2.3-fold more potent than improgan (EC50 = 199.5 nmol). As found previously with improgan, VUF5420-induced antinociception was unaffected by administration of the opioid antagonist naltrexone, but was inhibited by the CB1 antagonist SR141716A, suggesting a non-opioid, cannabinoid-related analgesic action. However, VUF5420 showed very low affinity (Kd approximately 10 microM) on CB1-receptor activation of 35S-GTPgammaS binding, indicating that this drug does not directly interact with the CB1 receptor in vivo. The present results show that VUF5420 is a high potency, improgan-like, non-opioid analgesic which may indirectly activate cannabinoid pain-relieving mechanisms.

Cannabinoid-improgan cross-tolerance: Improgan is a cannabinomimetic analgesic lacking affinity at the cannabinoid CB1 receptor.

Improgan is a non-opioid analgesic which does not act at known histamine or cannabinoid receptors. Because improgan antinociception is blocked by low doses of a cannabinoid CB1 antagonist, the present experiments determined if development of cannabinoid tolerance in mice would alter improgan antinociception. Twice-daily injections of Delta9-tetrahydrocannabinol (THC, 10 mg/kg, s.c.) for 3.5 days induced 47-54% and 42-56% reductions in cannabinoid (WIN 55,212-2, 20 microg, i.c.v.) and improgan (30 microg, i.c.v.) antinociception, respectively, as compared with responses from vehicle-treated groups. Because improgan lacks cannabinoid-like side effects in rats, and does not act directly on cannabinoid CB1 receptors, the finding that development of cannabinoid tolerance reduces improgan antinociception suggests that this drug may release endocannabinoids, or activate novel cannabinoid sites. Either possibility offers the potential for developing new types of analgesics.

Antinociceptive, brain-penetrating derivatives related to improgan, a non-opioid analgesic.

The antinociceptive profile of selected histamine H(2) and histamine H(3) receptor antagonists led to the discovery of improgan, a non-brain-penetrating analgesic agent which does not act on known histamine receptors. Because no chemical congener of improgan has yet been discovered which has both antinociceptive and brain-penetrating properties, the present study investigated the antinociceptive effects of a series of chemical compounds related to zolantidine, a brain-penetrating histamine H(2) receptor antagonist. The drugs studied presently contain the piperidinomethylphenoxy (PMPO) moiety, hypothesized to introduce brain-penetrating characteristics. Following intracerebroventricular (i.c.v.) dosing in rats, six of eight drugs produced dose- and time-related antinociception on both the tail flick and hot plate tests over a nearly eight-fold range of potencies. Ataxia and other motor side effects were observed after high doses of these drugs, but two of the compounds (SKF94674 and loxtidine) produced maximal antinociception at doses which were completely devoid of these motor effects. Consistent with the hypothesis that PMPO-containing drugs are brain-penetrating analgesics, SKF94674 and another derivative (JB-9322) showed dose-dependent antinociceptive activity 15 to 30 min after systemic dosing in mice, but these effects were accompanied by seizures and death beginning 45 min after dosing. Other drugs showed a similar pattern of antinociceptive and toxic effects. In addition, loxtidine produced seizures without antinociception, whereas zolantidine produced neither effect after systemic dosing in mice. Although several of the drugs tested have histamine H(2) receptor antagonist activity, neither the antinociception nor the toxicity was correlated with histamine H(2) receptor activity. The present results are the first to demonstrate the existence of brain-penetrating antinociceptive agents chemically related to zolantidine and improgan, but further studies are needed to understand the mechanisms of both the pain relief and toxicity produced by these agents.

Neuronal cytochrome P450 activity and opioid analgesia: relevant sites and mechanisms.

Recent studies suggest a functional role for neuronal cytochrome P450 monooxygenase (P450) activity in opioid analgesia. To characterize the relevant receptors, brain areas, and circuits, detailed in vitro and in vivo studies were performed with the highly selective µ opioid receptor agonist DAMGO in neuronal P450-deficient mutant (Null) and control mice. Homogenates of brain regions and spinal cord showed no differences in DAMGO-induced activation of [(35)S]- GTPγS binding between Null and control mice, indicating no genotype differences in µ opioid receptor signaling, receptor affinities or receptor densities. Intracerebroventricular (icv) DAMGO produced robust, near-maximal, analgesic responses in control mice which were attenuated by 50% in Null mice, confirming a role for µ opioid receptors in activating P450-associated responses. Intra-periaqueductal gray (PAG) and intra-rostral ventromedial medulla (RVM) injections of DAMGO revealed deficits in Null (vs. control) analgesic responses, yet no such genotype differences were observed after intrathecal DAMGO administration. Taken with earlier published findings, the present results suggest that activation of µ opioid receptors in both the PAG and in the RVM relieves pain by mechanisms which include nerve-terminal P450 enzymes within inhibitory PAG-RVM projections. Spinal opioid analgesia, however, does not seem to require such P450 enzyme activity.

Histamine-induced modulation of nociceptive responses.

Because previous studies suggest an antinociceptive role for the neuromodulator histamine (HA) in the periaqueductal grey or the nearby dorsal raphe (PAG/DR), a detailed pharmacological investigation of the effects of intracerebral HA on the hot-plate nociceptive test was performed in rats. Intracerebral microinjections of HA (1 microgram) into the PAG/DR or into the median raphe evoked a mild, reversible antinociceptive response; injections into lateral or dorsal midbrain evoked either a delayed response or no response, respectively. In the PAG/DR, the HA dose-response curve had an inverted U-shape, showing that HA can induce both antinociceptive (0.3-3 micrograms) and pro-nociceptive (10-30 micrograms) responses. Larger doses of HA (e.g., 100 micrograms) produced irreversible and highly variable antinociceptive responses that were accompanied by behavioral and histopathological changes; such effects, indicative of toxicity, were not observed after 0.3 microgram of HA, the peak antinociceptive dose. HA (0.3 microgram) antinociception was completely inhibited by intracerebral co-administration of the opiate antagonist naloxone (1 ng), the H1-receptor antagonist temelastine (20 pg), and the H2-receptor antagonist tiotidine (1 ng); none of these drugs altered nociceptive scores in the absence of HA. These results show that: (1) HA, a neurotransmitter in the PAG, can evoke antinociception in the absence of other behavioral or toxic effects; and (2) HA antinociception depends on the activation of both opiate and HA receptors in the PAG/DR.(ABSTRACT TRUNCATED AT 250 WORDS)

Improgan antinociception does not require neuronal histamine or histamine receptors.

Improgan, a chemical congener of the H(2) antagonist cimetidine, induces antinociception following intracerebroventricular (i.c.v.) administration in rodents, but the mechanism of action of this compound remains unknown. Because the chemical structure of improgan closely resembles those of histamine and certain histamine blockers, and because neuronal histamine is known to participate in pain-relieving responses, the antinociceptive actions of improgan were evaluated in mice containing null mutations in the genes for three histamine receptors (H(1), H(2), and H(3)) and also in the gene for histidine decarboxylase (the histamine biosynthetic enzyme). Similar to earlier findings in Swiss-Webster mice, improgan induced maximal, reversible, dose-related reductions in thermal nociceptive responses in ICR mice, but neither pre-improgan (baseline) nor post-improgan nociceptive latencies were changed in any of the mutant mice as compared with wild-type controls. Improgan also had weak inhibitory activity in vitro (pK(i)=4.7-4.9) on specific binding to three recently-discovered, recombinant isoforms of the rat H(3) receptor (H(3A), H(3B), and H(3C)). The present findings strongly support the hypothesis that neuronal histamine and its receptors fail to play a role in improgan-induced antinociception.

Activation of spinal histamine H3 receptors inhibits mechanical nociception.

Previous studies have suggested a possible pain-modulatory role for histamine H(3) receptors, but the localization of these receptors and nature of this modulation is not clear. In order to explore the role of spinal histamine H(3) receptors in the inhibition of nociception, the effects of systemically (subcutaneous, s.c.) and intrathecally (i.t.) administered histamine H(3) receptor agonists were studied in rats and mice. Immepip (5 mg/kg, s.c.) produced robust antinociception in rats on a mechanical (tail pinch) test but did not alter nociceptive responses on a thermal (tail flick) test. In contrast, this treatment in mice (immepip, 5 and 30 mg/kg, s.c.) did not change either mechanically or thermally evoked nociceptive responses. When administered directly into the spinal subarachnoid space, immepip (15-50 microg, i.t.) and R-alpha-methylhistamine (50 microg, i.t.) had no effect in rats on the tail flick and hot plate tests, but produced a dose- and time-dependent inhibition (90-100%) of nociceptive responses on the tail pinch test. This attenuation was blocked by administration of thioperamide (10 mg/kg, s.c.), a histamine H(3) receptor antagonist. Intrathecally administered thioperamide also reversed antinociceptive responses induced by systemically administered immepip, which demonstrates a spinal site of action for the histamine H(3) receptor agonist. In addition, intrathecally administered immepip (25 microg) produced maximal antinociception on the tail pinch test in wild type, but not in histamine H(3) receptor knockout (H(3)KO) mice. These findings demonstrate an antinociceptive role for spinal histamine H(3) receptors. Further studies are needed to confirm the existence of modality-specific (i.e. mechanical vs. thermal) inhibition of nociception by these receptors, and to assess the efficacy of spinally delivered histamine H(3) receptor agonists for the treatment for pain.

Significance of neuronal cytochrome P450 activity in opioid-mediated stress-induced analgesia.

Stressful environmental changes can suppress nociceptive transmission, a phenomenon known as "stress-induced analgesia". Depending on the stressor and the subject, opioid or non-opioid mechanisms are activated. Brain µ opioid receptors mediate analgesia evoked either by exogenous agents (e.g. morphine), or by the release of endogenous opioids following stressful procedures. Recent work with morphine and neuronal cytochrome P450 (P450)-deficient mice proposed a signal transduction role for P450 enzymes in µ analgesia. Since µ opioid receptors also mediate some forms of stress-induced analgesia, the present studies assessed the significance of brain P450 activity in opioid-mediated stress-induced analgesia. Two widely-used models of opioid stress-induced analgesia (restraint and warm water swim) were studied in both sexes of wild-type control and P450-deficient (Null) mice. In control mice, both stressors evoked moderate analgesic responses which were blocked by pretreatment with the opioid antagonist naltrexone, confirming the opioid nature of these responses. Consistent with literature, sex differences (control female>control male) were seen in swim-induced, but not restraint-induced, analgesia. Null mice showed differential responses to the two stress paradigms. As compared with control subjects, Null mice showed highly attenuated restraint-induced analgesia, showing a critical role for neuronal P450s in this response. However, warm water swim-induced analgesia was unchanged in Null vs. control mice. Additional control experiments confirmed the absence of morphine analgesia in Null mice. These results are the first to show that some forms of opioid-mediated stress-induced analgesia require brain neuronal P450 activity.

Deficits in neuronal cytochrome P450 activity attenuate opioid analgesia but not opioid side effects.

Morphine-like analgesics act on µ opioid receptors in the CNS to produce highly effective pain relief, but the same class of receptors also mediates non-therapeutic side effects. The analgesic properties of morphine were recently shown to require the activity of a brain neuronal cytochrome P450 epoxygenase, but the significance of this pathway for opioid side effects is unknown. Here we show that brain P450 activity is not required for three of morphine׳s major side effects (respiratory depression, constipation, and locomotor stimulation). Following systemic or intracerebroventricular administration of morphine, transgenic mice with brain neuron - specific reductions in P450 activity showed highly attenuated analgesic responses as compared with wild-type (control) mice. However, brain P450-deficient mice showed normal morphine-induced side effects (respiratory depression, locomotor stimulation, and inhibition of intestinal motility). Pretreatment of control mice with the P450 inhibitor CC12 similarly reduced the analgesia, but not these side effects of morphine. Because activation of brain µ opioid receptors produces both opioid analgesia and opioid side effects, dissociation of the mechanisms for the therapeutic and therapy-limiting effects of opioids has important consequences for the development of analgesics with reduced side effects and/or limited addiction liability.